Method of identifying sequence variants using concatenation

ABSTRACT

Described herein, among other things, is a method of sequencing, comprising: concatenating a plurality of fragments of genomic DNA to produce concatenated DNA; sequencing the concatenated DNA to produce a plurality of sequence reads, wherein at least some of the sequence reads comprise: at least the sequence of the 3′ and/or 5′ ends of a fragment that corresponds to the locus of interest and sequence of one or both of the fragments that flank the fragment in the concatenated DNA; and grouping the sequence reads that corresponds to the locus of interest using, for each of the grouped sequence reads: the 3′ and/or 5′ end sequences; and/or the flanking sequence.

CROSS-REFERENCING

This application is a continuation of U.S. application Ser. No.15/756,051, filed on Feb. 27, 2018, which is a § 371 national phase ofInternational Application No. PCT/IB2016/055252, filed on Sep. 1, 2016,which claims the benefit of UK patent application serial no. 1515558.3,filed on Sep. 2, 2015, which applications are incorporated by referenceherein.

BACKGROUND

Many diseases are caused by somatic mutations. Because somatic mutationsonly occur in a fraction of the cells in the body, they can be difficultto detect with high sensitivity and specificity by next generationsequencing. One problem is that every library preparation method andsequencing platform results in sequence reads that contain errors, e.g.,PCR errors and sequencing errors. While it is sometimes possible tocorrect systematic errors (e.g., those that are correlated with knownparameters including sequencing cycle-number, strand, sequence-contextand base substitution probabilities), it is often impossible to figureout with any certainty whether a variation in a sequence is caused by anerror or if it is a “real” mutation. This problem is exacerbated insamples in which the sample has low diversity and/or mutation-containingpolynucleotides are present only at relatively low levels, e.g., lessthan 5%, in the sample. For example, if a sample contains only one copyof a mutation-containing polynucleotide in a background of hundreds ofpolynucleotides that are otherwise identical to the mutation-containingpolynucleotide except that they do not contain the mutation, then, afterthose polynucleotides have been sequenced, it is often impossible totell whether the variation (which may only be observed in about 1/100 ofthe sequence reads) is an error that occurred during amplification orsequencing. Thus, the detection of somatic mutations that cause diseasescan be extremely difficult to detect with any certainty.

SUMMARY

Described herein, among other things, is a method of sequencing,comprising: concatenating a plurality of fragments of genomic DNA toproduce concatenated DNA; sequencing fragments of the concatenated DNA,or amplification products thereof, to produce a plurality of sequencereads, wherein at least some of the sequence reads comprise: at leastthe sequence of the 3′ and/or 5′ ends of a fragment that corresponds tothe locus of interest; and sequence of one or both of the fragments thatflank the fragment in the concatenated DNA; and grouping the sequencereads that corresponds to the locus of interest using, for each of thegrouped sequence reads: the 3′ and/or 5′ end sequences; and/or theflanking sequence.

As will be described in greater detail below (and illustrated in FIG.1), the method can be used to ascertain if a sequence variation isgenuine. In these embodiments, the method may further comprise:determining which groups of sequence reads contain a potential sequencevariation for the locus of interest; and calculating a probability thatthe potential sequence variation is a genuine mutation or an artifactusing: (i) the number of reads in the group that contain the potentialsequence variation, (ii) the number of groups that contain the potentialsequence variation, and (iii) the total number of groups correspondingto the locus of interest.

The method finds particular use in analyzing samples in which DNA haslimited diversity and that also contain fragments having a low copynumber mutation (e.g., a sequence caused by a mutation that is presentat low copy number relative to sequences that do not contain themutation), which are both features of many patient samples that can beobtained non-invasively, such as circulating, cell-free DNA (e.g.,ctDNA) samples, which can be obtained from peripheral blood, orinvasively, e.g., tissue sections. In such samples, the mutant sequencesmay only be present at a very limited copy number (e.g., less than 10,less than 5 copies or even 1 copy in a background of hundreds orthousands of copies of the wild type sequence) and there is a highprobability that at least some of the mutant fragments have an otherwiseidentical sequence (including identical end sequence(s)) to a wild typefragment. In these situations, it can be very difficult to identify asequence variation with significant confidence.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to scale. Indeed, the dimensions of the variousfeatures are arbitrarily expanded or reduced for clarity. Included inthe drawings are the following figures.

FIG. 1 schematically illustrates some of the principles of the presentmethod. Note that there are two A molecules (with identical 5′/3′breakpoints) shown by dashed lines. The fragments are concatenated. Thetwo A molecules have different flanking fragments. The concatemers arethen amplified or, optionally, fragmented and amplified. The light greysequences are adaptors. After sequencing reads can be grouped accordingto 5′/3′ breakpoints and the flanking sequences. This allows both Asequences to be individually identified.

FIG. 2 shows a flow chart illustrating an exemplary bioinformaticsworkflow. The first three steps can in theory be performed in any order.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are described.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

Numeric ranges are inclusive of the numbers defining the range. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention. Accordingly, the terms definedimmediately below are more fully defined by reference to thespecification as a whole.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely”,“only” and the like in connection with the recitation of claim elements,or the use of a “negative” limitation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

The term “sample” as used herein relates to a material or mixture ofmaterials, typically containing one or more analytes of interest. In oneembodiment, the term as used in its broadest sense, refers to any plant,animal or viral material containing DNA or RNA, such as, for example,tissue or fluid isolated from an individual (including withoutlimitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva andtissue sections) or from in vitro cell culture constituents, as well assamples from the environment.

The term “nucleic acid sample,” as used herein, denotes a samplecontaining nucleic acids. Nucleic acid samples used herein may becomplex in that they contain multiple different molecules that containsequences. Genomic DNA samples from a mammal (e.g., mouse or human) aretypes of complex samples. Complex samples may have more than about 10⁴,10⁵, 10⁶ or 10⁷, 10⁸, 10⁹ or 10¹⁰ different nucleic acid molecules. ADNA target may originate from any source such as genomic DNA, or anartificial DNA construct. Any sample containing nucleic acid, e.g.,genomic DNA from tissue culture cells or a sample of tissue, may beemployed herein.

The term “mixture” as used herein, refers to a combination of elements,that are interspersed and not in any particular order. A mixture isheterogeneous and not spatially separable into its differentconstituents. Examples of mixtures of elements include a number ofdifferent elements that are dissolved in the same aqueous solution and anumber of different elements attached to a solid support at randompositions (i.e., in no particular order). A mixture is not addressable.To illustrate by example, an array of spatially separated surface-boundpolynucleotides, as is commonly known in the art, is not a mixture ofsurface-bound polynucleotides because the species of surface-boundpolynucleotides are spatially distinct and the array is addressable.

The term “nucleotide” is intended to include those moieties that containnot only the known purine and pyrimidine bases, but also otherheterocyclic bases that have been modified. Such modifications includemethylated purines or pyrimidines, acylated purines or pyrimidines,alkylated riboses or other heterocycles. In addition, the term“nucleotide” includes those moieties that contain hapten or fluorescentlabels and may contain not only conventional ribose and deoxyribosesugars, but other sugars as well. Modified nucleosides or nucleotidesalso include modifications on the sugar moiety, e.g., wherein one ormore of the hydroxyl groups are replaced with halogen atoms or aliphaticgroups, or are functionalized as ethers, amines, or the like.

The term “nucleic acid” and “polynucleotide” are used interchangeablyherein to describe a polymer of any length, e.g., greater than about 2bases, greater than about 10 bases, greater than about 100 bases,greater than about 500 bases, greater than 1000 bases, greater than10,000 bases, greater than 100,000 bases, greater than about 1,000,000,up to about 10¹⁰ or more bases composed of nucleotides, e.g.,deoxyribonucleotides or ribonucleotides, and may be producedenzymatically or synthetically (e.g., PNA as described in U.S. Pat. No.5,948,902 and the references cited therein) which can hybridize withnaturally occurring nucleic acids in a sequence specific manneranalogous to that of two naturally occurring nucleic acids, e.g., canparticipate in Watson-Crick base pairing interactions.Naturally-occurring nucleotides include guanine, cytosine, adenine,thymine, uracil (G, C, A, T and U respectively). DNA and RNA have adeoxyribose and ribose sugar backbone, respectively, whereas PNA'sbackbone is composed of repeating N-(2-aminoethyl)-glycine units linkedby peptide bonds. In PNA various purine and pyrimidine bases are linkedto the backbone by methylenecarbonyl bonds. A locked nucleic acid (LNA),often referred to as inaccessible RNA, is a modified RNA nucleotide. Theribose moiety of an LNA nucleotide is modified with an extra bridgeconnecting the 2′ oxygen and 4′ carbon. The bridge “locks” the ribose inthe 3′-endo (North) conformation, which is often found in the A-formduplexes. LNA nucleotides can be mixed with DNA or RNA residues in theoligonucleotide whenever desired. The term “unstructured nucleic acid,”or “UNA,” is a nucleic acid containing non-natural nucleotides that bindto each other with reduced stability. For example, an unstructurednucleic acid may contain a G′ residue and a C′ residue, where theseresidues correspond to non-naturally occurring forms, i.e., analogs, ofG and C that base pair with each other with reduced stability, butretain an ability to base pair with naturally occurring C and Gresidues, respectively. Unstructured nucleic acid is described inUS20050233340, which is incorporated by reference herein for disclosureof UNA.

The term “oligonucleotide” as used herein denotes a single-strandedmultimer of nucleotide of from about 2 to 200 nucleotides, up to 500nucleotides in length. Oligonucleotides may be synthetic or may be madeenzymatically, and, in some embodiments, are 30 to 150 nucleotides inlength. Oligonucleotides may contain ribonucleotide monomers (i.e., maybe oligoribonucleotides) or deoxyribonucleotide monomers, or bothribonucleotide monomers and deoxyribonucleotide monomers. Anoligonucleotide may be 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60,61 to 70, 71 to 80, 81 to 100, 101 to 150 or 151 to 200 nucleotides inlength, for example.

“Primer” means an oligonucleotide, either natural or synthetic that iscapable, upon forming a duplex with a polynucleotide template, of actingas a point of initiation of nucleic acid synthesis and being extendedfrom its 3′ end along the template so that an extended duplex is formed.The sequence of nucleotides added during the extension process isdetermined by the sequence of the template polynucleotide. Usuallyprimers are extended by a DNA polymerase. Primers are generally of alength compatible with their use in synthesis of primer extensionproducts, and are usually in the range of between 8 to 100 nucleotidesin length, such as 10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21to 50, 22 to 45, 25 to 40, and so on. Typical primers can be in therange of between 10-50 nucleotides long, such as 15-45, 18-40, 20-30,21-25 and so on, and any length between the stated ranges. In someembodiments, the primers are usually not more than about 10, 12, 15, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or70 nucleotides in length.

Primers are usually single-stranded for maximum efficiency inamplification, but may alternatively be double-stranded or partiallydouble-stranded. If double-stranded, the primer is usually first treatedto separate its strands before being used to prepare extension products.This denaturation step is typically effected by heat, but mayalternatively be carried out using alkali, followed by neutralization.Also included in this definition are toehold exchange primers, asdescribed in Zhang et al (Nature Chemistry 2012 4: 208-214), which isincorporated by reference herein.

Thus, a “primer” is complementary to a template, and complexes byhydrogen bonding or hybridization with the template to give aprimer/template complex for initiation of synthesis by a polymerase,which is extended by the addition of covalently bonded bases linked atits 3′ end complementary to the template in the process of DNAsynthesis.

The term “hybridization” or “hybridizes” refers to a process in which aregion of nucleic acid strand anneals to and forms a stable duplex,either a homoduplex or a heteroduplex, under normal hybridizationconditions with a second complementary nucleic acid strand, and does notform a stable duplex with unrelated nucleic acid molecules under thesame normal hybridization conditions. The formation of a duplex isaccomplished by annealing two complementary nucleic acid strand regionsin a hybridization reaction. The hybridization reaction can be made tobe highly specific by adjustment of the hybridization conditions (oftenreferred to as hybridization stringency) under which the hybridizationreaction takes place, such that two nucleic acid strands will not form astable duplex, e.g., a duplex that retains a region ofdouble-strandedness under normal stringency conditions, unless the twonucleic acid strands contain a certain number of nucleotides in specificsequences which are substantially or completely complementary. “Normalhybridization or normal stringency conditions” are readily determinedfor any given hybridization reaction. See, for example, Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NewYork, or Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press. As used herein, the term “hybridizing”or “hybridization” refers to any process by which a strand of nucleicacid binds with a complementary strand through base pairing.

A nucleic acid is considered to be “selectively hybridizable” to areference nucleic acid sequence if the two sequences specificallyhybridize to one another under moderate to high stringency hybridizationand wash conditions. Moderate and high stringency hybridizationconditions are known (see, e.g., Ausubel, et al., Short Protocols inMolecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold SpringHarbor, N.Y.). One example of high stringency conditions includehybridization at about 42° C. in 50% formamide, 5×SSC, 5×Denhardt'ssolution, 0.5% SDS and 100 μg/ml denatured carrier DNA followed bywashing two times in 2×SSC and 0.5% SDS at room temperature and twoadditional times in 0.1×SSC and 0.5% SDS at 42° C.

The term “duplex,” or “duplexed,” as used herein, describes twocomplementary polynucleotide region that are base-paired, i.e.,hybridized together.

The term “amplifying” as used herein refers to the process ofsynthesizing nucleic acid molecules that are complementary to one orboth strands of a template nucleic acid. Amplifying a nucleic acidmolecule may include denaturing the template nucleic acid, annealingprimers to the template nucleic acid at a temperature that is below themelting temperatures of the primers, and enzymatically elongating fromthe primers to generate an amplification product. The denaturing,annealing and elongating steps each can be performed one or more times.In certain cases, the denaturing, annealing and elongating steps areperformed multiple times such that the amount of amplification productis increasing, often times exponentially, although exponentialamplification is not required by the present methods. Amplificationtypically requires the presence of deoxyribonucleoside triphosphates, aDNA polymerase enzyme and an appropriate buffer and/or co-factors foroptimal activity of the polymerase enzyme. The term “amplificationproduct” refers to the nucleic acids, which are produced from theamplifying process as defined herein. DNA can be amplified by isothermalamplification methods or by PCR for example. In some embodiments, DNAcan be amplified by a whole genome amplification (WGA method).

The terms “determining,” “measuring,” “evaluating,” “assessing,”“assaying,” and “analyzing” are used interchangeably herein to refer toany form of measurement, and include determining if an element ispresent or not. These terms include both quantitative and/or qualitativedeterminations. Assessing may be relative or absolute. “Assessing thepresence of” includes determining the amount of something present, aswell as determining whether it is present or absent.

The term “copies of fragments” refers to the product of amplification,where a copy of a fragment can be a reverse complement of a strand of afragment, or have the same sequence as a strand of a fragment.

The term “substantially identical sequences” refers to sequences thatare at least 95% or at least 99% identical to one another.

The term “using” has its conventional meaning, and, as such, meansemploying, e.g., putting into service, a method or composition to attainan end. For example, if a program is used to create a file, a program isexecuted to make a file, the file usually being the output of theprogram. In another example, if a computer file is used, it is usuallyaccessed, read, and the information stored in the file employed toattain an end. Similarly if a unique identifier, e.g., a barcode isused, the unique identifier is usually read to identify, for example, anobject or file associated with the unique identifier.

A “plurality” contains at least 2 members. In certain cases, a pluralitymay have at least 2, at least 5, at least 10, at least 100, at least100, at least 10,000, at least 100,000, at least 10⁶, at least 10⁷, atleast 10⁸ or at least 10⁹ or more members.

If two nucleic acids are “complementary,” they hybridize with oneanother under high stringency conditions. The term “perfectlycomplementary” is used to describe a duplex in which each base of one ofthe nucleic acids base pairs with a complementary nucleotide in theother nucleic acid. In many cases, two sequences that are complementaryhave at least 10, e.g., at least 12 or 15 nucleotides ofcomplementarity.

An “oligonucleotide binding site” refers to a site to which anoligonucleotide hybridizes in a target polynucleotide. If anoligonucleotide “provides” a binding site for a primer, then the primermay hybridize to that oligonucleotide or its complement.

The term “strand” as used herein refers to a nucleic acid made up ofnucleotides covalently linked together by covalent bonds, e.g.,phosphodiester bonds. In a cell, DNA usually exists in a double-strandedform, and as such, has two complementary strands of nucleic acidreferred to herein as the “top” and “bottom” strands. In certain cases,complementary strands of a chromosomal region may be referred to as“plus” and “minus” strands, the “first” and “second” strands, the“coding” and “noncoding” strands, the “Watson” and “Crick” strands orthe “sense” and “antisense” strands. The assignment of a strand as beinga top or bottom strand is arbitrary and does not imply any particularorientation, function or structure. The nucleotide sequences of thefirst strand of several exemplary mammalian chromosomal regions (e.g.,BACs, assemblies, chromosomes, etc.) is known, and may be found inNCBI's Genbank database, for example.

The term “top strand,” as used herein, refers to either strand of anucleic acid but not both strands of a nucleic acid. When anoligonucleotide or a primer binds or anneals “only to a top strand,” itbinds to only one strand but not the other. The term “bottom strand,” asused herein, refers to the strand that is complementary to the “topstrand.” When an oligonucleotide binds or anneals “only to one strand,”it binds to only one strand, e.g., the first or second strand, but notthe other strand.

The term “extending”, as used herein, refers to the extension of aprimer by the addition of nucleotides using a polymerase. If a primerthat is annealed to a nucleic acid is extended, the nucleic acid acts asa template for extension reaction.

The terms “A-tailed”, “C-tailed”, “G-tailed”, and “T-tailed”, as usedherein, refer to fragments with single-base 3′ overhangs. If addedenzymatically, typically these overhangs are generated via thenon-template addition by a polymerase of a single nucleotide to the 3′end of a blunt fragment, but other methods may also be used.Alternatively 3′ overhangs may be generated when annealing twooligonucleotides.

The term “sequencing,” as used herein, refers to a method by which theidentity of at least 10 consecutive nucleotides (e.g., the identity ofat least 20, at least 50, at least 100 or at least 200 or moreconsecutive nucleotides) of a polynucleotide is obtained.

The terms “next-generation sequencing” or “high-throughput sequencing”,as used herein, refer to the so-called parallelizedsequencing-by-synthesis or sequencing-by-ligation platforms currentlyemployed by Illumina, Life Technologies, and Roche, etc. Next-generationsequencing methods may also include nanopore sequencing methods such asthose commercialized by Oxford Nanopore Technologies,electronic-detection based methods such as Ion Torrent technologycommercialized by Life Technologies, or single-moleculefluorescence-based methods commercialized by Pacific Biosciences.

The term “asymmetric adaptor”, as used herein, refers to an adaptorthat, when ligated to both ends of a double stranded nucleic acidfragment, will lead to a top strand that contains a 5′ tag sequence thatis not the same as or complementary to the tag sequence at the 3′ end.Exemplary asymmetric adapters are described in: U.S. Pat. Nos. 5,712,126and 6,372,434 and WO/2009/032167; all of which are incorporated byreference herein in their entirety. An asymmetrically tagged fragmentcan be amplified by two primers: one that hybridizes to a first tagsequence added to the 3′ end of a strand, and another that hybridizes tothe complement of a second tag sequence added to the 5′ end of a strand.Y-adaptors and hairpin adaptors (which can be cleaved, after ligation,to produce a “Y-adaptor”) are examples of asymmetric adaptors.

The term “Y-adaptor” refers to an adaptor that contains: adouble-stranded region and a single-stranded region in which theopposing sequences are not complementary. The end of the double-strandedregion can be joined to target molecules such as double-strandedfragments of genomic DNA, e.g., by ligation or a transposase-catalyzedreaction. Each strand of an adaptor-tagged double-stranded DNA that hasbeen ligated to a Y adaptor is asymmetrically tagged in that it has thesequence of one strand of the Y-adaptor at one end and the other strandof the Y-adaptor at the other end. Amplification of nucleic acidmolecules that have been joined to Y-adaptors at both ends results in anasymmetrically tagged nucleic acid, i.e., a nucleic acid that has a 5′end containing one tag sequence and a 3′ end that has another tagsequence.

The term “hairpin adaptor” refers to an adaptor that is in the form of ahairpin. In one embodiment, after ligation the hairpin loop can becleaved to produce strands that have non-complementary tags on the ends.In some cases, the loop of a hairpin adaptor may contain a uracilresidue, and the loop can be cleaved using uracil DNA glycosylase andendonuclease VIII, although other methods are known.

The term “tagging” as used herein, refers to the appending of a sequencetag (that contains an identifier sequence) onto a nucleic acid molecule.A sequence tag may be added to the 5′ end, the 3′ end, or both ends ofnucleic acid molecule. A sequence tag can be added to a fragment byligating an oligonucleotide to the fragment.

The terms “identifier sequence” and “tag sequence that identifies” areused interchangeably herein to, refer to a sequence of nucleotides usedto identify and/or track the source of a polynucleotide in a reaction.After the polynucleotides in a sample are sequenced, the identifiersequence can be used to distinguish the sequence reads and/or determinefrom which sample a sequence read is derived. An “identifier sequence”may be referred to a “sample barcode”, “index” or “indexer” sequence inother publications. For example, different samples (e.g.,polynucleotides derived from different individuals, different tissues orcells, or polynucleotides isolated at different times points), can betagged with identifier sequences that are different from one anotherand, after the samples are tagged, they are pooled. After sequencing,the source of a sequence can be tracked back to a particular sampleusing the identifier sequence. Identifier sequences can be added to asample by ligation, by primer extension using a tailed primer thatcontains an identifier sequence in a 5′ tail, or using a transposon. Anidentifier sequence can range in length from 2 to 100 nucleotide basesor more and may include multiple subunits, where each differentidentifier has a distinct identity and/or order of subunits. A sampleidentifier sequence may be added to the 5′ end of a polynucleotide orthe 3′ end of a polynucleotide, for example. In particular embodiments,a barcode sequence may have a length in range of from 1 to 36nucleotides, e.g., from 6 to 30 nucleotides, or 8 to 20 nucleotides. Incertain cases, the molecular identifier sequence may beerror-correcting, meaning that even if there is an error (e.g., if thesequence of the molecular barcode is mis-synthesized, mis-read or isdistorted by virtue of the various processing steps leading up to thedetermination of the molecular barcode sequence) then the code can stillbe interpreted correctly. Descriptions of exemplary error correctingsequences can be found throughout the literature (e.g., US20100323348and US20090105959, which are both incorporated herein by reference). Insome embodiments, an identifier sequence may be of relatively lowcomplexity (e.g., may be composed of a mixture of 8 to 1024 differentsequences), although higher complexity identifier sequences can be usedin some cases.

The term “sample identifier sequence” is a sequence of nucleotides thatis appended to a target polynucleotide, where the sequence identifiesthe sample (e.g., which individual, which cell, which tissue, or whichtimes points, etc.) from which a sequence read is derived. In use, eachsample is tagged with a different sample identifier sequence (e.g., onesequence is appended to each sample, where the different samples areappended to different sequences), and the tagged samples can be pooled.After the samples are sequenced, the sample identifier sequence can beused to identify the source of the sequences.

As used herein, the term “complementary” in the context of sequencereads that are complementary, refers to reads for sequences that, afterthe sequences have been trimmed to remove adaptor sequences, aresubstantially complementary to one another and, in some cases, haveidentical or near identical ends, indicating that the reads are derivedfrom the same initial template molecules.

The term “identical or near-identical sequences”, as used herein, refersto near duplicate sequences, as measured by a similarity function,including but not limited to a Hamming distance, Levenshtein distance,Jaccard distance, cosine distance etc. (see, generally, Kemena et al,Bioinformatics 2009 25: 2455-65). The exact threshold depends on theerror rate of the sample preparation and sequencing used to perform theanalysis, with higher error rates requiring lower thresholds ofsimilarity. In certain cases, substantially identical sequences have atleast 98% or at least 99% sequence identity.

The term “fragmentation breakpoint” is intended to refer to the site atwhich a nucleic acid is cleaved to produce a fragment. Two fragmentsthat have the same fragmentation breakpoints have the same sequences attheir ends (excluding any exogenous sequences that have been added tothe fragments). Fragmentation breakpoints can be generated by random ornon-random methods. In analyzing sequence reads, the fragmentationbreakpoint may be identified as the boundary between genomic-derivedsequence and adaptor derived sequences (including or excluding anyoverhangs in adaptor sequences).

The term “identical or near-identical fragmentation breakpoints”, asused herein, refers to two molecules that have the same 5′ end, the same3′ end, or the same 5′ and 3′ ends, where the differences in sequenceare due to a PCR error, a sequencing error or a mutation. Afragmentation breakpoint can be determined by removing non-targetsequences from a sequence read, leaving the sequence of the target. Thefirst nucleotide of the trimmed sequence represents the first nucleotideafter the fragmentation breakpoint. In sequencing an amplified sample,two sequence reads that correspond to fragments that have identical ornear-identical fragmentation breakpoints can be derived from the sameinitial fragment. In many cases, 8-30 nucleotides at the end of atrimmed sequence can be compared to the ends of other trimmed sequencesto determine if the fragmentation breakpoints are the same or different.In many cases, fragmentation breakpoints can be identified after mappingreads to a reference sequence. Fragmentation breakpoints may be mappedusing, e.g., Picard MarkDuplicates (available from the BroadInstitute)), Samtools rmdup (see, e.g., Li et al. Bioinformatics 2009,25: 2078-2079) and BioBamBam (Tischler et al, Source Code for Biologyand Medicine 2014, 9:13).

The term “pooling”, as used herein, refers to the combining, e.g.,mixing, of two samples such that the molecules within those samplesbecome interspersed with one another in solution.

The term “pooled sample”, as used herein, refers to the product ofpooling.

The term “target enrichment”, as used herein, refers to a method inwhich selected sequences are separated from other sequences in a sample.This may be done by hybridization to a probe, e.g., hybridizing abiotinylated oligonucleotide to the sample to produce duplexes betweenthe oligonucleotide and the target sequence, immobilizing the duplexesvia the biotin group, washing the immobilized duplexes, and thenreleasing the target sequences from the oligonucleotides. Alternatively,a selected sequence may be enriched by amplifying that sequence, e.g.,by PCR using one or more primers that hybridize to a site that isproximal to the target sequence.

The terms “minority variant” and “sequence variation”, as used herein,is a variant that is present a frequency of less than 50%, relative toother molecules in the sample. In some cases, a minority variant may bea first allele of a polymorphic target sequence, where, in a sample, theratio of molecules that contain the first allele of the polymorphictarget sequence compared to molecules that contain other alleles of thepolymorphic target sequence is 1:100 or less, 1:1,000 or less, 1:10,000or less, 1:100,000 or less or 1:1,000,000 or less.

The term “concatenating”, as used herein, refers to the joining offragments to one another in a random order and orientation to produce aconcatenation product, i.e., single molecule in which the initialfragments, or copies thereof, are covalently linked to one another,either directly or indirectly.

The term “concatenated DNA”, as used herein, refers to a product ofconcatenating fragments of DNA to one another. Such a molecule maycontain at least 3, at least 5, at least 10, at least 50, at least 100,at least 500, or at least 1000 fragments that are joined to one another,either directly or indirectly (e.g., via a junction adaptor). Aconcatenated molecule may be linear or circular. DNA fragments may beconcatenated by ligation or overlap extension, for example.

The term “ligating”, as used herein, refers to the enzymaticallycatalyzed joining of the terminal nucleotide at the 5′ end of a firstDNA molecule to the terminal nucleotide at the 3′ end of a second DNAmolecule such that the first DNA molecule and the second DNA moleculebecome covalently linked to one another, either directly or indirectly(e.g., via an intervening sequence such as a junction adaptor).

The term “ligated to one another via junction adaptor”, as used herein,refers to the indirect ligation of two or more DNA molecules to oneanother via an adaptor. A concatenated DNA molecule that containsfragments that have been ligated to one another via a junction adaptormay contain a first fragment, an adaptor, a second fragment, an adaptor,a third fragment, an adaptor, and so on.

The term “overlap extension”, as used herein, refers to a way forconcatenating DNA fragments together by primer extension. In someembodiments, overlap extension may comprise ligating adaptors onto theends of the fragments, and then making a concatemer of the adaptorligated fragments using primers that have 5′ tails that hybridize to oneanother (see, e.g., Horton BioTechniques, Vol. 54, No. 3, March 2013,pp. 129-133) or by designing the adaptors so that they hybridize to oneanother, and then extending the adaptors using another fragment as atemplate.

The term “potential sequence variation”, as used herein, refers to asequence variation, e.g., a substitution, deletion, insertion orrearrangement of one or more nucleotides in one sequence relative toanother that could potentially be present in the original, unamplifiedsample. A sequence variation may be a genuine sequence variation or anartifact.

The term “genuine sequence variation”, as used herein, refers to asequence variation that is present in the original, unamplified sample.A genuine sequence variation may be a SNP, or it may be a somatic orgermline mutation.

The term “artifact”, as used herein, refers to a sequence variation thatresulted from an amplification error (i.e., a mis-incorporation of baseby the polymerase during amplification) or a miss-call of a base duringsequencing. Neither of these mutations are present in the original,unamplified sample and, as such, they are referred to as “artifacts”.

As used herein, the term “correspond to”, with reference to a sequenceread that corresponds to a locus of interest, refers to a sequence readobtained from an amplification product of that locus.

The term “at least the sequence of the 3′ and/or 5′ ends of a fragment”,as used herein, refers to the 3′ and/or 5′ sequences that are at theends of the fragment, i.e., immediately adjacent to the fragmentationbreakpoint of the fragment. A sequence read that contains at least thesequence of the 3′ and/or 5′ ends of a fragment may contain at least 10,at least 20, at least 30, at least 50, at least 100 bases at the 3′ endof a fragment, and/or at least 10, at least 20, at least 30, at least 50or at least 100 bases at the 5′ end of a fragment. In some embodiments,one sequence read may contain the entire contiguous sequence of one ormore fragments, in which case the read may contain sequence from bothends of the fragment. In some cases, the sequence may be a paired endsequence, in which case sequence read may contain only the ends of afragment or, if the paired end reads are overlapping, the sequence ofthe entire fragment, including the ends.

The term “flanks”, as used herein in the context of a fragment thatflanks a fragment of interest, refers to a fragment that is immediatelyadjacent to the fragment of interest (excluding any adaptor or exogenoussequence that is present between the first and second fragments).

The term “flanking sequence”, as used herein, refers to sequence that isobtained from a fragment that flanks a fragment of interest. In somecases, the flanking sequence may be obtained from the end of the secondfragment that is joined to the fragment of interest (e.g., at least 10,at least 20, at least 30, at least 50, at least 100 bases at the 3′ endor 5′ end of the flanking fragment).

The term “whole genome amplification”, as used herein, refers to anytype of amplification reaction that results in a relatively uniformamplification of substantially all template sequences in a sample (e.g.,at least 90% or 95% of the template sequences). Exemplary whole genomeamplification methods include degenerate oligonucleotide PCR (DOP-PCR),primer extension preamplification (PEP), and adapter-linker PCR. Wholegenome amplification methods of particular interest include multipledisplacement amplification (“MDA”; see Dean et al Proc. Natl. Acad. Sci.2002 99: 5261-5266 and Nelson Biotechniques 2002 Suppl: 44-47), as wellas multiple annealing and looping based amplification cycles (“MalBac”;see Zong et al Science. 2012 338:1622-1626) and PicoPLEX, which bothinvolve a limited MDA-based pre-amplification followed by PCR (see,e.g., de Bourcy et al, PLoS One. 2014 9: e105585; Arneson et al Oncol.2012: 710692 and Mohlendick et al, Curr Protoc Cell Biol. 2014 65:1-22).

The term “sequence diversity”, as used herein, refers to the number of5′ and/or 3′ breakpoints that are associated with a plurality offragments corresponding to a target sequence.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anucleic acid” includes a plurality of such nucleic acids and referenceto “the compound” includes reference to one or more compounds andequivalents thereof known to those skilled in the art, and so forth.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, N.Y., Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, A., Principles of Biochemistry3^(rd) Ed., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W. H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Some of the principles of the method are shown in FIG. 1. With referenceto FIG. 1, the method can be initiated by concatenating a plurality offragments of genomic DNA to produce concatenated DNA. In this method,the fragments may have a median size that is below 1 kb (e.g., in therange of 50 bp to 500 bp, or 80 bp to 400 bp), although fragmentsoutside of this size range may be used in certain embodiments. Thefragments may be concatenated by ligation (e.g., by ligating thefragments directly to one another, or by ligating the fragments to oneanother via a junction adaptor, such as a double-stranded adaptor of8-50 bp) or, alternatively, by overlap extension (e.g., by ligating thefragments an adaptor and concatenating the adapter-ligated fragments byoverlap extension). As would be apparent, in some embodiments, the endsof the fragments may be blunted first if the fragments are going to bedirectly ligated to one another. In embodiments in which the fragmentsare ligated to one another via a junction adaptor, the fragments may beoptionally A-tailed prior to ligation to an adaptor with single base 3′T overhangs. Alternatively, the fragments may be T-tailed and ligated toadaptors with A overhangs, C-tailed and ligated to adaptors with Goverhangs, or G-tailed and ligated to adaptors with C overhangs, etc.Each of these combinations of overhangs is designed to ensure thatadaptors can only ligate to fragments (and vice versa) and thatadaptor-adaptor ligation and fragment-fragment ligation cannot occur. Asa result, the concatemer formed will consist of repeatedfragment-adaptor units, facilitating breakpoint recognition whenanalyzing the sequencing data. In certain embodiments, a first portionof a sample may be treated to add a single nucleotide overhang (e.g., anA), and a second portion of a sample may be treated to add a singlenucleotide overhang that is complementary to the first portion of thesample (e.g., a T). After this treatment, the two portions of the samplecan be combined and ligated to one another to produce a concatemer inwhich the fragments from the first portion (which may be tailed with anA, for example) alternate with the fragments from the second portion(which may be talked with a T, for example).

The concatenated DNA may comprise at least a thousand or at least tenthousand concatenated DNA molecules, and each molecule may contain atleast 3, at least 5, at least 10, at least 50, at least 100, at least500, or at least 1000 fragments that are joined one another in a randomorder and orientation.

Next, the method comprises sequencing fragments of the concatenated DNA,or amplification products thereof, to produce a plurality of sequencereads. In some embodiments, the concatenated DNA may be sequenceddirectly (i.e., without amplification). In these embodiments, theconcatenated DNA may be fragmented to a desired length (e.g., a medianlength that is 50 nt to 1000 nt or 100 nt 500 nt longer than the lengthof the initial fragments), ligating the fragmentation products toadaptors (where these steps may be done separately or by tagmentation,see, e.g., Caruccio, Methods Mol. Biol. 2011; 733:241-55), andsequencing the ligation products directly. In other embodiments (and asshown in FIG. 1) the method may comprise amplifying the concatenated DNAbetween the concatenation and sequencing steps. In some embodiments, theamplification may be done by fragmenting the concatenated DNA to adesirable size (e.g., a median length that is 50-500 nt longer than thelength of the initial fragments), adding an adaptor (e.g., an asymmetricadaptor) to the fragments, and amplifying the fragments by PCR usingprimers that hybridize to the adaptor sequences. As noted above, thefragmentation and adaptor ligation steps may be mediated by atransposase (see, e.g., Caruccio, Methods Mol. Biol. 2011; 733:241-55),in which case the steps may be done simultaneously, i.e., in the samereaction using a process that is often referred to as “tagmentation”. Inother embodiments, the fragmenting may be done mechanically (e.g., bysonication, nebulization, or shearing) or using a double stranded DNA“dsDNA” fragmentase enzyme (New England Biolabs, Ipswich Mass.). In someof these methods (e.g., the mechanical and fragmentase methods), afterthe DNA is fragmented, the ends are polished and A-tailed prior toligation to the adaptor. Alternatively, the ends may be polished andligated to adaptors in a blunt-end ligation reaction. In someembodiments, the concatenated DNA may be amplified using a whole genomeamplification method (e.g., MDA, MALBAC or PicoPLEX), which methods canbe tailored to produce “sequence ready” amplicons of a desired length(e.g., amplicons that have sequencing-compatible adaptor sequences attheir ends). The sequenced fragments, or amplification products thereof,may have a median length in the range of 200 bp to 2 kb, or, a lengththat is below 1 kb such as in the range of 50 bp to 500 bp, or 80 bp to400 bp, although fragments having a median size outside of this rangemay be used.

In some embodiments, fragments are attached to a generic asymmetricadaptor before amplification thereby allowing the identification ofsequencing reads that derive from either the top or bottom strand of adouble-stranded fragment. This approach can be used in error-correctionbased on the idea that for a true DNA mutation, complementarysubstitutions should be present on both strands and, as such, a mutationcan only be called with confidence if it is present in sequences fromboth strands.

As would be apparent, the primers used for amplification may becompatible with use in any next generation sequencing platform in whichprimer extension is used, e.g., Illumina's reversible terminator method,Roche's pyrosequencing method (454), Life Technologies' sequencing byligation (the SOLiD platform), Life Technologies' Ion Torrent platformor Pacific Biosciences' fluorescent base-cleavage method. Examples ofsuch methods are described in the following references: Margulies et al.(Nature 2005 437: 376-80); Ronaghi et al. (Analytical Biochemistry 1996242: 84-9); Shendure et al. (Science 2005 309: 1728); Imelfort et al.(Brief Bioinform. 2009 10:609-18); Fox et al. (Methods Mol Biol. 2009;553:79-108); Appleby et al. (Methods Mol Biol. 2009; 513:19-39) Englishet al. (PLoS One. 2012 7: e47768) and Morozova and Marra (Genomics. 200892:255-64), which are incorporated by reference for the generaldescriptions of the methods and the particular steps of the methods,including all starting products, reagents, and final products for eachof the steps.

The sequencing step may be done using any convenient next generationsequencing method and may result in at least 10,000, at least 50,000, atleast 100,000, at least 500,000, at least 1M at least 10M at least 100Mor at least 1B sequence reads. In some cases, the reads are paired-endreads.

As shown in FIG. 1, at least some of the sequence reads comprise: (i) atleast the sequence of the 3′ and/or 5′ ends of a fragment thatcorresponds to the locus of interest (which correspond to the fragment'sbreakpoints); and (ii) sequence of one or both of the fragments thatflank that fragment in the concatenated DNA. Imagine two differentmolecules with the same (i) 5′/3′ fragmentation breakpoints. The twomolecules can be disambiguated if concatenated to one or more different(ii) flanking fragments. In some cases, the sequence corresponding tothe fragment that flanks the fragment of the locus of interest may bethe sequence of the end of the flanking fragment (i.e., adjacent to thebreakpoint of the fragment) and, as such, the identity of the fragmentthat flanks the fragment of the locus of interest in the concatenatedDNA may be determined using a sequence of at least 10, at least 20 or atleast 30 nucleotides that is close to the breakpoint of that fragment.As noted above, in some cases, the initial DNA fragments may beconcatenated to one another via a junction adaptor, in which thebreakpoints of the fragments may be readily identified by identifyingthe junction adaptor sequence. In embodiments in which the initial DNAfragments are concatenated to one another directly, the breakpoints ofthe fragments may be identified by comparing the sequence reads to areference genome and, if a first part of a sequence read maps to onelocation in the genome and a second part of the sequence (which isadjacent to the first part) read maps to another location in the genome,then the breakpoint is between the first and second parts.

The sequence reads may be processed and grouped in any convenient way.In some implementations, initial processing of the sequence reads mayinclude identification of molecular barcodes (including sampleidentifier sequences or sub-sample identifier sequences), and/ortrimming reads to remove low quality or adaptor sequences. In addition,quality assessment metrics can be run to ensure that the dataset is ofan acceptable quality. After initial processing and as shown in FIG. 2,the sequence reads may be grouped by grouping the sequence reads thatcorresponds to the locus of interest using, for each of the groupedsequence reads: (i) the 3′ and/or 5′ end sequences of a fragmentcorresponding to a locus of interest; and/or (ii) a sequence that flanksthe sequences of a fragment corresponding to a locus of interest, i.e.,the identity of the “flanking sequence”. In other words, the sequencereads that correspond to a locus of interest may be grouped by a) one orboth of the fragmentation breakpoints of the fragment corresponding tothe locus of interest and/or b) the identity of one or more of thefragments that were joined to the fragment corresponding to locus ofinterest in the concatenation step.

Assuming that the initial fragments (i.e., the fragments that wereconcatenated) were made by fragmenting a more intact sample in a randomor semi-random fashion, different fragments having the same sequence canbe distinguished by their fragmentation breakpoint. As such, groupingthe sequence reads by their fragmentation breakpoints provides a way todetermine if a particular sequence (e.g., a sequence variant) is presentin more than one starting molecule. For example, if two groups ofsequence reads that have different fragmentation breakpoints contain asequence variation, then one can be more confident that the sequencevariation is genuine than if the sequence variation is only present in asingle group. Further, because the concatenation process is random,different molecules that correspond to a locus of interest can bedistinguished by the fragments that they are ligated to. For example iftwo fragments correspond to a locus of interest are otherwise identicalin sequence and have identical fragmentation breakpoints but are flankedby different fragments, then those molecules can be distinguished in thesequence reads.

As such, the method may be used to determine whether a potentialsequence variation is a genuine mutation or an artifact. This method maycomprise, after grouping: determining which groups of sequence readscontain a potential sequence variation for the locus of interest; andcalculating a probability that the potential sequence variation is agenuine mutation or an artifact using: (i) the number of reads in thegroup that contain the potential sequence variation, (ii) the number ofgroups that contain the potential sequence variation, and (iii) thetotal number of groups corresponding to the locus of interest. Thiscalculating step may incorporate an error model that estimates thelikelihood that a given sequencing and/or PCR error might occur. Severalsources of information can be used in this assessment including (1) abase transition probability matrix (Statistical Methods inBioinformatics: An Introduction by Warren J. Ewens, Gregory R. Gran). Inone embodiment, the matrix can be estimated during consensus sequencederivation. For example, the number of reads at a given position in aread group that either agree or disagree with the most likely consensusbase can be used to estimate transition probabilities. (2) The sequencecontext of a variant, e.g. whether the variant is adjacent to ahomopolymer run; (3) known sequencer error modalities, such assequencing cycle number; and (4) performance characteristics ofsequencing positive and/or negative controls.

As noted above, the confidence that a potential sequence variation is agenuine sequence variation (rather than a PCR or sequencing error)increases if it is represented in more than one group, particularlygroups that are defined by a larger number of sequence reads.

In some embodiments, the nucleic acids sequenced in the sequencing stepmay be enriched by target enrichment, many methods for which are known.In some embodiments, the enrichment may be done by hybridization to aprobe, e.g., by SURESELECT™, which may involve hybridizing theamplification products to an oligonucleotide (e.g., RNA) probe thatcontains an affinity tag (e.g., biotin) to the amplification products.The resultant duplexes can be separated from other molecules' productsby binding the oligonucleotide to a solid support and washing, and thetarget molecules can be released. Target enrichment can also be doneusing target-specific primers, by PCR amplification (see, e.g.,US20130231253).

In some embodiments, the amplicons sequenced in the sequencing step maybe selected by size (e.g., AMPure XP beads) so that shorter amplicons(of 200-300 bp) can be sequenced.

In some embodiments, sample identifiers (i.e., a sequence thatidentifies the sample to which the sequence is added, which can identifythe patient, or a tissue, etc.) can be added to the polynucleotidesprior to sequencing, so that multiple (e.g., at least 2, at least 4, atleast 8, at least 16, at least 48, at least 96 or more) samples can bemultiplexed. In these embodiments, the sample identifier can be presentin an adaptor added after fragmentation of the concatenated DNA, forexample. In some embodiments, the adaptor may comprise a sampleidentifier sequence that identifies the sample to which the adaptor isadded.

The method described above can be employed to analyze genomic DNA fromvirtually any organism, including, but not limited to, plants, animals(e.g., reptiles, mammals, insects, worms, fish, etc.), tissue samples,bacteria, fungi (e.g., yeast), phage, viruses, cadaveric tissue,archaeological/ancient samples, etc. In certain embodiments, the genomicDNA used in the method may be derived from a mammal, wherein certainembodiments the mammal is a human. In exemplary embodiments, the samplemay contain genomic DNA from a mammalian cell, such as, a human, mouse,rat, or monkey cell. The sample may be made from cultured cells or cellsof a clinical sample, e.g., a tissue biopsy, scrape or lavage or cellsof a forensic sample (i.e., cells of a sample collected at a crimescene). In particular embodiments, the fragments of DNA may be obtainedfrom a biological sample such as cells, tissues, bodily fluids, andstool. Bodily fluids of interest include but are not limited to, blood,serum, plasma, saliva, mucous, phlegm, cerebral spinal fluid, pleuralfluid, tears, lactal duct fluid, lymph, sputum, cerebrospinal fluid,synovial fluid, urine, amniotic fluid, and semen. In particularembodiments, a DNA may be obtained from a subject, e.g., a human. Insome embodiments, the sample analyzed may be a sample of cell-free“circulating” DNA (e.g., circulating tumor DNA) obtained from peripheralblood, e.g., from the blood of a patient or of a pregnant female. Insome embodiments, the DNA comprises fragments of human genomic DNA. Insome embodiments, the DNA may be obtained from a cancer patient. In someembodiments, the DNA may be made by extracting fragmented DNA from apatient sample, e.g., a formalin-fixed paraffin embedded tissue sample.In some embodiments, the patient sample may be cell-free DNA from abodily fluid, e.g., peripheral blood. The DNA fragments used in theinitial step of the method should be non-amplified DNA that has not beendenatured beforehand.

The DNA in the initial sample may be made by extracting genomic DNA froma biological sample, and then fragmenting it. The fragments in theinitial sample may have a median size that is below 1 kb (e.g., in therange of 50 bp to 500 bp, or 80 bp to 400 bp), although fragments havinga median size outside of this range may be used. In other embodiments,the DNA in the initial sample may already be fragmented (e.g., as is thecase for FPET samples and ctDNA).

In some embodiments, the amount of DNA in a sample may be limiting. Forexample, the initial sample of fragmented DNA may contain less than 200ng of fragmented human DNA, e.g., 10 pg to 200 ng, 100 pg to 200 ng, 1ng to 200 ng or 50 ng to 50 ng, or less than 10,000 (e.g., less than5,000, less than 1,000, less than 500, less than 100 or less than 10)haploid genome equivalents, depending on the genome.

Bioinformatics Workflow

One implementation of an exemplary bioinformatics workflow is shown inFIG. 2.

The sequence reads may be analyzed by a computer and, as such,instructions for performing the steps set forth below may be set forthas programing that may be recorded in a suitable physical computerreadable storage medium. The general principles of some of the analysissteps are described below.

The informatics steps of the above-described method can be implementedon a computer. In certain embodiments, a general-purpose computer can beconfigured to a functional arrangement for the methods and programsdisclosed herein. The hardware architecture of such a computer is wellknown by a person skilled in the art, and can comprise hardwarecomponents including one or more processors (CPU), a random-accessmemory (RAM), a read-only memory (ROM), an internal or external datastorage medium (e.g., hard disk drive). A computer system can alsocomprise one or more graphic boards for processing and outputtinggraphical information to display means. The above components can besuitably interconnected via a bus inside the computer. The computer canfurther comprise suitable interfaces for communicating withgeneral-purpose external components such as a monitor, keyboard, mouse,network, etc. In some embodiments, the computer can be capable ofparallel processing or can be part of a network configured for parallelor distributive computing to increase the processing power for thepresent methods and programs. In some embodiments, the program code readout from the storage medium can be written into memory provided in anexpanded board inserted in the computer, or an expanded unit connectedto the computer, and a CPU or the like provided in the expanded board orexpanded unit can actually perform a part or all of the operationsaccording to the instructions of the program code, so as to accomplishthe functions described below. In other embodiments, the method can beperformed using a cloud computing system. In these embodiments, the datafiles and the programming can be exported to a cloud computer that runsthe program and returns an output to the user.

A system can, in certain embodiments, comprise a computer that includes:a) a central processing unit; b) a main non-volatile storage drive,which can include one or more hard drives, for storing software anddata, where the storage drive is controlled by disk controller; c) asystem memory, e.g., high speed random-access memory (RAM), for storingsystem control programs, data, and application programs, includingprograms and data loaded from non-volatile storage drive; system memorycan also include read-only memory (ROM); d) a user interface, includingone or more input or output devices, such as a mouse, a keypad, and adisplay; e) an optional network interface card for connecting to anywired or wireless communication network, e.g., a printer; and f) aninternal bus for interconnecting the aforementioned elements of thesystem.

The memory of a computer system can be any device that can storeinformation for retrieval by a processor, and can include magnetic oroptical devices, or solid state memory devices (such as volatile ornon-volatile RAM). A memory or memory unit can have more than onephysical memory device of the same or different types (for example, amemory can have multiple memory devices such as multiple drives, cards,or multiple solid state memory devices or some combination of the same).With respect to computer readable media, “permanent memory” refers tomemory that is permanent. Permanent memory is not erased by terminationof the electrical supply to a computer or processor. Computer hard-driveROM (i.e., ROM not used as virtual memory), CD-ROM, floppy disk and DVDare all examples of permanent memory. Random Access Memory (RAM) is anexample of non-permanent (i.e., volatile) memory. A file in permanentmemory can be editable and re-writable.

Operation of the computer is controlled primarily by an operatingsystem, which is executed by the central processing unit. The operatingsystem can be stored in a system memory. In some embodiments, theoperating system includes a file system. In addition to an operatingsystem, one possible implementation of the system memory includes avariety of programming files and data files for implementing the methoddescribed below. In certain cases, the programming can contain aprogram, where the program can be composed of various modules, and auser interface module that permits a user to manually select or changethe inputs to or the parameters used by the program. The data files caninclude various inputs for the program.

In certain embodiments, instructions in accordance with the methoddescribed herein can be coded onto a computer-readable medium in theform of “programming,” where the term “computer readable medium” as usedherein refers to any storage or transmission medium that participates inproviding instructions and/or data to a computer for execution and/orprocessing. Examples of storage media include a floppy disk, hard disk,optical disk, magneto-optical disk, CD-ROM, CD-R, magnetic tape,non-volatile memory card, ROM, DVD-ROM, Blue-ray disk, solid state disk,and network attached storage (NAS), whether or not such devices areinternal or external to the computer. A file containing information canbe “stored” on computer readable medium, where “storing” means recordinginformation such that it is accessible and retrievable at a later dateby a computer.

The computer-implemented method described herein can be executed usingprograms that can be written in one or more of any number of computerprogramming languages. Such languages include, for example, Java (SunMicrosystems, Inc., Santa Clara, Calif.), Visual Basic (Microsoft Corp.,Redmond, Wash.), and C++ (AT&T Corp., Bedminster, N.J.), as well as anymany others.

In any embodiment, data can be forwarded to a “remote location,” where“remote location,” means a location other than the location at which theprogram is executed. For example, a remote location could be anotherlocation (e.g., office, lab, etc.) in the same city, another location ina different city, another location in a different state, anotherlocation in a different country, etc. As such, when one item isindicated as being “remote” from another, what is meant is that the twoitems can be in the same room but separated, or at least in differentrooms or different buildings, and can be at least one mile, ten miles,or at least one hundred miles apart. “Communicating” informationreferences transmitting the data representing that information aselectrical signals over a suitable communication channel (e.g., aprivate or public network). “Forwarding” an item refers to any means ofgetting that item from one location to the next, whether by physicallytransporting that item or otherwise (where that is possible) andincludes, at least in the case of data, physically transporting a mediumcarrying the data or communicating the data. Examples of communicatingmedia include radio or infra-red transmission channels as well as anetwork connection to another computer or networked device, and theinternet or including email transmissions and information recorded onwebsites and the like.

Some embodiments include implementation on a single computer, or acrossa network of computers, or across networks of networks of computers, forexample, across a network cloud, across a local area network, onhand-held computer devices, etc. In certain embodiments, one or more ofthe steps described herein are implemented on a computer program(s).Such computer programs execute one or more of the steps describedherein. In some embodiments, implementations of the subject methodinclude various data structures, categories, and modifiers describedherein, encoded on computer-readable medium(s) and transmissible overcommunications network(s).

Software, web, internet, cloud, or other storage and computer networkimplementations of the present invention could be accomplished withstandard programming techniques to conduct the various assigning,calculating, identifying, scoring, accessing, generating or discardingsteps.

The following patent publications are incorporated by reference for allpurposes, particularly for methods by which nucleic acid molecules maybe manipulated, reagents for doing the same, for sequencing librarypreparation workflow, sequencing methods, data processing methods, andfor definitions of certain terms: U.S. Pat. No. 8,481,292, WO2013128281,and Casbon (Nuc. Acids Res. 2011, 22 e81), US20150044678, US20120122737,U.S. Pat. No. 8,476,018 and all references cited above and below.

Utility

As would be readily apparent, the method described above may be employedto analyze any type of sample, including, but not limited to samplesthat contain heritable mutations, samples that contain somaticmutations, samples from mosaic individuals, pregnant females (in whichsome of the sample contains DNA from a developing fetus), and samplesthat contain a mixture of DNA from different sources. In certainembodiments, the method may be used identify a minority variant that, insome cases, may be due to a somatic mutation in a person.

In some embodiments, the method may be employed to detect an oncogenicmutation (which may be a somatic mutation) in, e.g., PIK3CA, NRAS, KRAS,JAK2, HRAS, FGFR3, FGFR1, EGFR, CDK4, BRAF, RET, PGDFRA, KIT or ERBB2,which may be associated with breast cancer, melanoma, renal cancer,endometrial cancer, ovarian cancer, pancreatic cancer, leukemia,colorectal cancer, prostate cancer, mesothelioma, glioma,medullobastoma, polycythemia, lymphoma, sarcoma or multiple myeloma(see, e.g., Chial 2008 Proto-oncogenes to oncogenes to cancer. NatureEducation 1:1). Other oncogenic mutations (which may be somaticmutations) of interest include mutations in, e.g., APC, AXIN2, CDH1,GPC3, CYLD, EXT1, EXT2, PTCH, SUFU, FH, SDHB, SDHC, SDHD, VHL, TP53,WT1, STK11/LKB1, PTEN, TSC1, TSC2, CDKN2A, CDK4, RB1, NF1, BMPR1A, MEN1,SMAD4, BHD, HRPT2, NF2, MUTYH, ATM, BLM, BRCA1, BRCA2, FANCA, FANCC,FANCD2, FANCE, FANCF, FANCG, NBS1, RECQL4, WRN, MSH2, MLH1, MSH6, PMS2,XPA, XPC, ERCC2-5, DDB2 or MET, which may be associated with colon,thyroid, parathyroid, pituitary, islet cell, stomach, intestinal,embryonal, bone, renal, breast, brain, ovarian, pancreatic, uterine,eye, hair follicle, blood or uterus cancers, pilotrichomas,medulloblastomas, leiomyomas, paragangliomas, pheochromocytomas,hamartomas, gliomas, fibromas, neuromas, lymphomas or melanomas. In someembodiments, the method may be employed to detect a somatic mutation ingenes that are implicated in cancer, e.g., CTNNB1, BCL2, TNFRSF6/FAS,BAX, FBXW7/CDC4, GLI, HPVE6, MDM2, NOTCH1, AKT2, FOXO1A, FOXO3A, CCND1,HPVE7, TAL1, TFE3, ABL1, ALK, EPHB2, FES, FGFR2, FLT3, FLT4, KRAS2,NTRK1, NTRK3, PDGFB, PDGFRB, EWSR1, RUNX1, SMAD2, TGFBR1, TGFBR2, BCL6,EVI1, HMGA2, HOXA9, HOXA11, HOXA13, HOXC13, HOXD11, HOXD13, HOX11,HOX11L2, MAP2K4, MLL, MYC, MYCN, MYCL1, PTNP1, PTNP11, RARA, SS18 (see,e.g., Vogelstein and Kinzler 2004 Cancer genes and the pathways theycontrol. Nature Medicine 10:789-799). The method of embodiment may beemployed to detect any somatic mutation that is implicated in cancerwhich is catalogued by COSMIC (Catalogue of Somatic Mutations inCancer), data of which can be accessed on the internet.

Other mutations of interest include mutations in, e.g., ARID1A, ARID1B,SMARCA4, SMARCB1, SMARCE1, AKT1, ACTB/ACTG1, CHD7, ANKRD11, SETBP1,MLL2, ASXL1, which may be at least associated with rare syndromes suchas Coffin-Siris syndrome, Proteus syndrome, Baraitser-Winter syndrome,CHARGE syndrome, KBG syndrome, Schinzel-Giedion syndrome, Kabukisyndrome or Bohring-Opitz syndrome (see, e.g., Veltman and Brunner 2012De novo mutations in human genetic disease. Nature Reviews Genetics13:565-575). Hence, the method may be employed to detect a mutation inthose genes.

In other embodiments, the method may be employed to detect a mutation ingenes that are implicated in a variety of neurodevelopmental disorders,e.g., KAT6B, THRA, EZH2, SRCAP, CSF1R, TRPV3, DNMT1, EFTUD2, SMAD4,LIS1, DCX, which may be associated with Ohdo syndrome, hypothyroidism,Genitopatellar syndrome, Weaver syndrome, Floating-Harbor syndrome,hereditary diffuse leukoencephalopathy with spheroids, Olmsted syndrome,ADCA-DN (autosomal-dominant cerebellar ataxia, deafness and narcolepsy),mandibulofacial dysostosis with microcephaly or Myhre syndrome (see,e.g., Ku et al 2012 A new paradigm emerges from study of de novomutations in the context of neurodevelopmental disease. MolecularPsychiatry 18:141-153). The method may also be employed to detect asomatic mutation in genes that are implicated in a variety ofneurological and neurodegenerative disorders, e.g., SCN1A, MECP2,IKBKG/NEMO or PRNP (see, e.g., Poduri et al 2014 Somatic mutation,genetic variation, and neurological disease. Science 341(6141):1237758).

In some embodiments, a sample may be collected from a patient at a firstlocation, e.g., in a clinical setting such as in a hospital or at adoctor's office, and the sample may be forwarded to a second location,e.g., a laboratory where it is processed and the above-described methodis performed to generate a report. A “report” as described herein, is anelectronic or tangible document which includes report elements thatprovide test results that may indicate the presence and/or quantity ofminority variant(s) in the sample. Once generated, the report may beforwarded to another location (which may be the same location as thefirst location), where it may be interpreted by a health professional(e.g., a clinician, a laboratory technician, or a physician such as anoncologist, surgeon, pathologist or virologist), as part of a clinicaldecision.

EXAMPLES

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

Theoretical Background

Cell-free or circulating tumour DNA (ctDNA) is tumour DNA circulatingfreely in the blood of a cancer patient. Protocols to extract ctDNAgenerally aim to reduce contamination with normal DNA from leukocytes.This is achieved by rapid processing of whole blood by centrifugation toremove all cells, and analysis of the remaining plasma. ctDNA is highlyfragmented, with a mean fragment size ˜165 bp. Newman et al (Nat Med.2014 20: 548-54) made libraries from 7-32 ng ctDNA isolated from 1-5 mLplasma. This is equivalent to 2,121-9,697 haploid genomes (assuming 3.3pg per haploid genome). This range represents the maximum number ofunique molecules that can be captured and sequenced. In practice themaximum number of molecules that can be captured is reduced by randomfragmentation of regions covered by bait targets and inefficienciesduring library preparation and target recovery.

Different library preparation methods and next generation sequencing(NGS) chemistries have their own systematic error profiles. It ispossible to correct systematic errors, in part at least, that arecorrelated with known parameters including sequencing cycle-number,strand, sequence-context and base substitution probabilities (Meacham etal BMC Bioinformatics. 2011 12: 451). Random errors can be mitigated byreplicate sequencing. However, neither method is sufficient ifattempting to detect, with high specificity, a minority variant withfrequency of ≤˜3%. To improve error-detection and correction, severalgroups have used a repeat-code approach. The idea is to sequence copiesof the same molecule. The copies are then aligned and a majority-voteused to generate a consensus sequence, which removes most of the errors.In addition, differences between each copy and the consensus sequencescan be used to build up an error-model of a specific genomic region,which can then be applied to clean-up consensus sequences. For example,see Shugay et al. 2014 (Towards error-free profiling of immunerepertoires. Nature Methods 11, 653-655 (2014)).

Four main methods can be applied to identify copies of the samemolecule: (1) fragmentation breakpoints; (2) orientation of a DNA insertcompared to surrounding adaptor sequences; (3) DNA barcodes and (4)physical separation.

If, by chance, two molecules have the same 5′ and 3′ breakpoints thenone might incorrectly group reads and attempt to generate a consensussequence. This could result in a false negative if a variant base wasnot called as part of the consensus sequence (e.g. if there were agreater number of, or higher quality, reads including a non-variantbase). Additionally, an error model could be tricked into assuming thata variant base position was error-prone when in fact genuine calls aremixed from two, or more, different molecules. To mitigate this effectNewman et al. (2014) classified unique molecules as those with unique 5′and 3′ breakpoints and 100% sequence identity, ignoring low quality basecalls. This prevents false negative variants but is likely to increasefalse-positive calls owing to grouping of reads with errors. Newman etal. (2012) appear aware of this deficiency as they discuss implementingmolecular tagging approaches to improve data quality.

To estimate the frequency of molecules with identical 5′ and 3′breakpoints, one can make several simplifying assumptions: (1)breakpoints are randomly distributed; (2) to be captured a fragment musthave 100% match to an RNA bait; and (3) that the library has a fixedsized range between 120-165 bp. For example, imagine a fragment where a5′ breakpoint maps 25 bp upstream of the RNA bait. To be within thefixed library size range the 5′ breakpoint could be associated with anyof 20 different 3′ breakpoints. The same calculation can be performedfor each of the possible 5′ and 3′ breakpoints that generate fragmentswithin the size range. The total number of breakpoints can be calculatedusing: Σ_(d=0) ⁴⁵d+1 where d is the difference in length between themaximum fragment length and the RNA bait length. In the above example,d=45 giving 1,081 breakpoints. Next, we can estimate the number ofduplicate molecules using collision theory. The expected total number oftimes a selection will repeat a previous selection as x integers arechosen from a list of y integers (1, y) equals:

${x - y + {y\left( \frac{y - 1}{y} \right)}^{x}}.$In our case, this can be paraphrased as the expected total number oftimes a captured molecule x will have the same two breakpoints asanother captured molecule, where y is the number of molecules withdifferent breakpoints in the library. For example, if x=1,000 andy=1,081 then 347 captured molecules are expected to have the samebreakpoints as another captured molecule. In practice, the number ofmolecules that cannot be uniquely identified is likely higher than 347because some of the 1,081 breakpoint combinations are likely to beobserved more often than others, owing to the distribution of fragmentsizes around a mean length and biases in fragmentation breakpoints. Thissuggests that one needs information in addition to the fragmentationbreakpoints in order to uniquely identify molecules forerror-correction.

DNA barcodes can be used on their own, or in addition with fragmentationbreakpoints, to help identify duplicate molecules. There is an importantdistinction between methods where DNA barcodes are derived from adaptorsequences (made from oligonucleotides) and the case where DNA barcodesare derived from the random assortment and ligation of different genomicDNA sequences. If adaptor derived barcodes are attached en masse, theyare added to template DNA before amplification and great care must betaken to ensure that residual adaptors are removed before amplification.These restrictions are not necessary if using the current approach,where barcodes derive from genomic DNA sequences. In addition, adaptorbased tagging usually requires that the pool of barcode sequences arecarefully synthesized and/or pooled before tagging. If barcodes aredegenerate then there can be bias. If barcodes are pooled fromindividual synthesis reactions then care must be taken not to over- orunder-represent individual barcode sequences. This restriction does notoccur in the concatenation method where each fragment is randomlyassociated with two flanking fragments.

Physical separation methods include clonal amplification in amicrodroplet or on a solid-surface (e.g. an Illumina flow-cell).Microdroplet methods generally rely on limiting dilution of templatewhere each microdroplet contains only a fraction of the total genome(digital PCR, RainDance Technologies' Thunderstorm platform for targetenrichment or 10X Genomics GemCode system). Current physical methodsrequire complex microfluidics.

Example The “Concatanation” Approach

Patient plasma DNA is end-repaired to yield blunt-end fragments with5′-phosphates and 3′-hydroxyl termini Next, end-repaired fragments areligated into concatemers by T4 DNA ligase. Concatemers are likely to bea mixture of linear and circular molecules. Linear molecules are favoredin a small ligation volume (i.e. high DNA concentration). Ignoring thetermini of linear concatemers, each concatenated fragment is juxtaposedbetween two fragments from different regions of the genome. Because ofthe huge diversity of potential fragments in the human genome theprobability that two fragments have the same breakpoints and adjacentconcatemer fragments is small.

Concatemers are amplified by devised WGA methods, e.g., MALBAC orPicoPLEX, which use semi-degenerate primers and multiple linearextensions followed by PCR. The WGA methods can also tag amplicons withIllumina NGS adaptor sequences. ctDNA fragments have a mean length of˜165 base-pairs. The size distribution of amplicons generated byPicoPLEX DNA-Seq is 200-9,000 bp versus 400-1,900 for MALBAC WGA(excluding adaptors), however these methods may be tailored to producesmaller amplicons, as desired. The ideal insert size for someimplementations is ˜200-250 bp because this is likely to include the˜165 bp fragment of interest and limited sequence from 5′ and 3′adjacent fragments. Longer amplicons e.g. 1,000 bp are likely to includemultiple concatemer fragments. Longer amplicons are deleterious duringhybridization capture and NGS because: (1) they PCR with reducedefficiency; (2) a higher proportion of sequenced bases map off-target;and (3) a target positioned centrally in an amplicon might not becovered by paired-end reads. The simplest solution is to use a sizeselection (e.g. AMPure XP beads). Alternatively, PCR conditions could beoptimized for shorter ˜200-250 amplicons (e.g. by reducing the extensiontime during the thermocycling program).

In an alternative approach, the concatemers could be re-fragmented e.g.by sonication or transposon methods into shorter ˜200-250 bp fragmentsbefore amplification by PCR. However, this approach would lose some ofthe advantages of the linear extension cycles used in the WGA methods.

Amplicons can be used as template in an Agilent SureSelect hybridisationcapture. SureSelect RNA baits will capture both the ˜165 bp target butalso 35-85 bp from the two neighbouring fragments. After sequencing,custom scripts are used to identify: (1) on-target reads, (2)breakpoints between concatemers, and (3) adjacent concatemer sequences.Reads are then grouped using fragment 5′ and 3′ breakpoints in additionto the 5′ and 3′ sequences of adjacent fragments. Errors can becorrected using data from: (1) the estimated number of target moleculescaptured, (2) overlapping amplicons, (3) majority-vote consensussequences, and (4) error models derived from majority-vote consensussequences.

For ease of description, the method described in this section does notinclude adaptor ligation before concatenation. However, concatemerbreakpoints may be difficult to identify with sufficient sensitivity orin a reasonable time, and fragments that contain genomic breakpointsmight be difficult to identify unless multiple fragments are capturedand sequenced. To help identify these events, a constant region can beinserted between juxtaposed fragments in the concatemer. Junctions couldbe readily identified by, for example, using a sequence that does notnaturally exist in the genome being sequenced.

That which is claimed is:
 1. A method of sequencing, comprising: (a)concatenating a plurality of molecules of cfDNA (cell-free DNA) to eachother to produce concatenated cfDNA that comprises multiple molecules ofthe DNA joined together; (b) fragmenting the concatenated DNA to producefragments of concatenated DNA; and (c) sequencing the fragments ofconcatenated DNA, or amplification products thereof, to produce aplurality of sequence reads.
 2. The method of claim 1, furthercomprising: (d) grouping the sequence reads that correspond to a locusof interest using: one or more end sequences of a fragment of the locusof interest; or the flanking sequences of the fragment of the locus ofinterest, wherein sequence reads that are grouped together have the same3′ end, the same 5′ end and/or the same flaking sequences.
 3. The methodof claim 2, further comprising: (e) identifying a potential sequencevariation in the locus of interest by analysis of a group of sequencereads.
 4. The method of claim 2, wherein: step (d) further comprisesgrouping the sequence reads according to their orientation, whereinsequence reads that are derived from the top strand of a double-strandedmolecule in the sample (a) are grouped together and sequence reads thatare derived from the bottom strand of the same double-stranded moleculeare grouped together; and the method further comprises: identifyingpotential sequence variation in a group of sequence reads thatcorrespond to the top strand of a molecule; and determining if thepotential sequence variation is in any of the sequence reads thatcorrespond to the bottom strand of the same molecule.
 5. The method ofclaim 1, wherein, in (a), the plurality of molecules are concatenated byligation.
 6. The method of claim 1, wherein, in (a), the plurality ofmolecules are ligated to an adaptor and then concatenated by an overlapextension reaction that comprises overlapping the ligation products withone another.
 7. The method of claim 1, wherein, in (a) the plurality ofmolecules of cfDNA are ligated to one another via junction adaptors, toproduce the concatenated DNA.
 8. The method of claim 7, wherein theplurality of molecules are A-tailed and the junction adaptors areT-tailed.
 9. The method of claim 1, wherein the method comprises,between steps (a) and (b), amplifying the concatenated DNA.
 10. Themethod of claim 9, wherein the amplifying is done by a whole genomeamplification method.
 11. The method of claim 1, wherein the amplifyingis done by fragmenting the concatenated DNA, adding adaptors to thefragments of concatenated DNA, and then amplifying the fragments ofconcatenated DNA by PCR.
 12. The method of claim 11, wherein thefragmenting and adding adaptors is done by a transposase.
 13. The methodof claim 1, wherein the cDNA of (a) is obtained from a cancer patient.14. The method of claim 1, wherein the fragmenting of (b) is done bysonication, nebulization, or shearing.